Cefaclor (7-phenylglycylamido!-3-chloro-3-cephem-4-carboxylic acid) is an antibiotic of the cephalosporin class. Its antibiotic activity is effective against a range of bacteria including Streptococcus pyogenes, Escherichia coli, Diplococcus pneumoniae, Shigella sp., Klebsiella pneumoniae, Aerobacter aerogenes and Salmonella heidelberg. This antibiotic has been synthesized from parent compounds by synthetic organic techniques (see, e.g. U.S. Pat. Nos. 3,925,372 and 4,064,343). A common synthetic technique is to protect the 4-carboxylate by esterification, proceed by a series of steps to modify the 3 position so that a sole chloride atom is eventually covalently bound at that position, and then remove the ester protecting group from the carboxylate. In this manner a variety of cephalosporin antibiotics have been synthesized.
Another antibiotic in the cephalosporin family is cephalexin (7-phenylglycylamido!-3-methyl-3-cephem-4-carboxylic acid). This antibiotic compound differs from cefaclor by the substitution of a methyl for the chloride at the 3 position. The synthesis of cephalexin is more easily achieved than the synthesis of cefaclor. However, the usefulness of cefaclor as an antibiotic surpasses that of cephalexin. For these reasons, it would be desirable to easily convert cephalexin to cefaclor. Synthetic organic routes can be utilized but, when these synthetic schemes are invoked, several steps are required to achieve this conversion. A simple, one-step process would be more desirable. Certain microorganisms contain haloperoxidases that can halogenate a wide variety of organic compounds (Franssen, M. C. R. et al., Adv. Applied Microbiol. 37: 41-99 (1992)). At the present time, these haloperoxidases do not appear to have commercial application as peroxidases. However, their use as halogenating agents has been sought. Despite optimistic predictions for the use of chloroperoxidases and other halogenating enzymes in the production of particular chemicals, the potential for the use of the haloperoxidases for this purpose remains unrealized. The major obstacles to fulfillment of these predictions lie in the narrow pH range of operation for these enzymes, the use of high concentrations of H.sub.2 O.sub.2 which can be toxic to the source of the enzymes, and the short half-lives of the enzyme biocatalysts, to name a few.
Most haloperoxidases concomitantly convert a peroxide to water in the course of oxidizing the chloride. Following this process, an enzymatic addition reaction occurs. However, to convert cephalexin to cefaclor, a substitution reaction is required; specifically, the substitution of a chloride for a methyl group. It would be desirable to have an enzyme preparation that not only chlorinates an organic compound but also substitutes a chloride for a methyl group on the organic compound at the same time. It would be especially desirable to have an enzyme that performs this substitution reaction at the appropriate position on a cephalexin molecule, thereby producing cefaclor.